Inhibitors of HIV-1 integrase (IN) inhibitors represent the most recent anti-AIDS drugs. Merck's raltegravir (RAL) (October 2007) and Gilead's elvitegravir (EVG) (August 2012) were the first two IN inhibitors to be approved by the FDA. These agents are members of a class of drugs called IN strand transfer inhibitors (INSTIs), due to their ability to preferentially block the enzyme's strand transfer (ST) reaction as compared to the enzymes 3'-processing (3'-P) reaction. Treatment with RAL and EVG selects for resistant forms of HIV and there is considerable cross-resistance to these two drugs. GlaxoSmithKline's dolutegravir (DTG) was approved by the FDA in August of 2013 as a 2nd-generation. DTG to be highly effective against WT HIV-1, but DTG is much more potent against IN mutants that confer resistance to first generation INSTIs. However, mutations that confer resistance to DTG can be selected in viruses that carry RAL and DTG resistance mutations. Utilizing my laboratory's design and synthetic capabilities, we have teamed with pharmacologists (Dr. Yves Pommier, NCI), virologists (Dr. Hughes, NCI) and structural biologists (Dr. Cherepanov, London Research Institute) to develop new INSTs. In collaboration with Dr. Cherepanov we obtained co-crystal structures of our best inhibitors bound to the prototype foamy virus (PFV) intasome (multimeric integrase with DNA substrate and metal cofactor). We have developed bicyclic 7,8-dihydroxy-3,4-dihydroisoquinolin-1(2H)-one based HIV-1 integrase inhibitors. We examined the potency of DTG and five of our best of 4-amino-1-hydroxy-2-oxo-1,8-naphthyridine-containing inhibitors against broad panels of well-characterized INSTI-resistant single and double mutants, and against the INSTI-resistant triple mutants identified in the VIKING clinical trials. We found that two of our synthetic inhibitors and are more broadly effective against the panel of IN mutants than DTG, the current care standard. Separately, we have begun investigating inhibitors of the polymerase (Pol) and RNase H domains of HIV- 1 reverse transcriptase (RT). Both RT and IN are members of a superfamily of polynucleotidyl transferases that use divalent metal cofactors. RT has two catalytic centers, a DNA polymerase (Pol) and an RNase H that cleaves RNA if it is part of an RNA-DNA duplex. These two activities collaborate to produce, from single-stranded genomic RNA, a double-stranded DNA that is the substrate for IN. At this time, there are no FDA-approved HIV-1 RNase H inhibitors and it remains as one of the last unutilized targets for AIDS therapeutics. Collateral cytotoxicity is a significant limitation of current RNase H inhibitors and achieving potent inhibition without concomitant cytotoxicity remains a major challenge to be addressed. Similar to the active site of IN, the active sites for both the Pol and the RNase H domains of RT have two bound Mg2+ ions and utilizing metal chelation may allow us to develop new classes of RT inhibitors directed at the Pol and RNase H active sites. This leaves open the possibility of developing metal-chelating inhibitors of Pol catalysis that may function in multiple ways. This idea is supported by the fact that metal chelation underlies the activity of INSTIs. In addition, there are metal-chelating RNase H inhibitors, although the compounds described in the literature have limited potency, and most are relatively toxic. As a consequence, no RNase H inhibitors have been approved by the FDA. We are exploring the use of metal-chelating molecules to inhibit RNase H as well as the Pol domains of RT. The original design of our metal-chelating INSTIs was derived from pyridopyrimidinone-containing motifs present in RNase H inhibitors developed by Merck. We have shown that structurally-related compounds inhibit both the Pol and RNase H activities of RT in biochemical assays and that the potency of these compounds is not affected, in a viral replication assay, by several mutations in RTthat affect the potency of non-nucleoside reverse transcriptase inhibitors (NNRTIs). The relatively low cytotoxicity of the compounds is notable, because many of the RNase H inhibitors that have been described in the literature show significant toxicity. Based on our promising early results, we prepared derivatives that lack the moiety that specifically interacts with the viral DNA at the IN active site. Our intent was to obtain compounds that would retain affinity at either the polymerase or the RNase H active sites but would decrease affinity at the IN active site. We tested three compounds for their ability to inhibit the RNase H and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 angstroms) of one of the compounds showed that it chelates the two magnesium ions at the RNase H active site; this prevents the RNase H active site from interacting with, and cleaving, the RNA strand of an RNA/DNA heteroduplex. All three compounds inhibited the polymerase-independent RNase H activity of HIV-1 RT. The compounds also reduced the polymerase activity of RT. Time of addition experiments showed that the compounds were more potent if they bind to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the PPT RNA primer that initiates the second strand of viral DNA synthesis. Although it is not a simple matter to correlate data from in vitro biochemical assays with efficacy against the virus, it appears that the potency of our best polymerase inhibitor is within an order of magnitude of nevirapine, an approved non-nucleoside RT inhibitor (NNRTI) in an in vitro assay. Experiments showed that this ability was a result of the compounds binding to the RNase H active site. These compounds appear to be relatively specific; they do not inhibit either E. coli RNase HI or human RNase H2. The compounds inhibit the replication of a HIV-1 based vector in a one-round assay. We have preliminary data showing that the antiviral potencies of the most potent compounds are not significantly affected by the G140S/Q148H double mutation in IN, which is frequently associated with INSTI resistance, nor by a panel of RT mutants (V106A, L100I/K103N, Y188L, Y181C, M184V, or K70R), which are broadly associated with resistance to NNRTIs. Importantly, the best of these compounds shows considerable potency (IC50 of 200 nM) with only modest toxicity in sensitive cell-based assay that detects changes in the ATP level in the cells [therapeutic index (TI) 70 as compared with Merck's reference RNase inhibitor (TI = 4)]. This suggests that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds.